The luminal surfaces of endothelial cells (ECs) that line our vasculature are coated with a glycocalyx of membrane-bound macromolecules comprised of sulfated proteoglycans, hyaluronic acid, sialic acids, glycocproteins and plasma proteins that adhere to this surface matrix. The endothelial glycocalyx layer (EGL) provides a multifunctional coating to the vasculature that is degraded in disease states such as atherosclerosis and diabetes. Because of dehydration artifacts associated with conventional electron microscopy, even such rudimentary characteristics as the thickness of the layer have not been firmly established. In vivo and in vitro studies have, however, shown that heparan sulfate proteoglycans mediate endothelial remodeling (cell elongation and alignment) in response to fluid shear stress and along with hyaluronic acid control vital mechanotransduction events such as fluid shear-induced stimulation of nitric oxide production, but the core proteins that are involved in these characteristic responses are not known. To address these fundamental questions that are crucial for our understanding of vascular function in health and disease, we will pursue the following studies in the proposed research: To elucidate the structure of the endothelial glycocalyx layer (EGL) we will apply, for the first time, cryo-transmission electron microscopy (cryo-TEM) in conjunction with confocal microscopy to determine its thickness and organization. To determine the proteoglycan core proteins that mediate EC remodeling and mechanotransduction in response to fluid shear stress we will use glycosaminoglycan (GAG) degrading enzymes, RNA interference technology and adhesion blocking amino acid sequences in vitro and knockout animals in vivo to deconstruct these processes. To carry out the projects in this Bioengineering Research Grant (BRG), we have organized a research team with core expertise in bioengineering including: in vitro shear experiments (Tarbell), and in vivo shear experiments (Fu) that is complemented by expertise in microscopy and molecular biology (Spray). PUBLIC HEALTH RELEVANCE: The research is important to public health because the endothelial glycocalyx layer (EGL) provides a multifunctional coating to the vasculature that is degraded in disease states such as atherosclerosis and diabetes. Degradation of the EGL leads to vasoregulatory dysfunction through, for example, loss of blood flow-induced stimulation of the potent vasodilator, nitric oxide. Knowledge of EGL structure and the core proteins and glycosaminoglycans involved in mechanotransduction/remodeling will be required if methods are to be developed to re-constitute or reinforce the EGL. A re-constituted EGL will restore critical vasoregulatory functions, thus combating disease. The foundational work proposed in this application is essential for translational work that will follow as part of the long range goals of this project.